Ejaculates were collected by artificial vagina from 3 Holstein sires and sorted to 90% purity for X-chromosome-bearing spermatozoa (range 88 to 93%) using flow cytometry. Sorted sperm were diluted to 2.1, 3.5, or 5.0 x 10(6) sperm per dose in an egg yolk (20%), Tris, glycerol (7%) extender. Collections were repeated until >600 straws per sperm dose per sire were obtained. Each sperm dose was loaded into color-coded 0.25-mL French straws, with alternate colors used to define treatments across sires. Within sires, straws were packaged at 9 per cane (3 of each color) and strategically allocated to 75 Holstein herds with targets for 50% use in heifers and 50% in lactating cows. Straw color was recorded in the on-farm record-keeping system at the time of insemination. Data were analyzed separately for cows and heifers. Among heifers, a total of 2,125 usable records were retrieved from 51 herds (238 +/- 5.5 services/ sperm dose per sire, range: 218 to 263). Conception rates in heifers were influenced by the sire x sperm dosage interaction. Within sire A, conception rates of heifers were greater for the 5 x 10(6) (59.5%) than for the 2.1 x 10(6) (46.4%) sperm dose and intermediate for the 3.5 x 10(6) sperm dose (52.2%). However, across sires, sperm dosage had no effect on heifer conception rates (46.7, 51.2, and 52.5% for the 2.1, 3.5, and 5.0 x 10(6) sperm dosages, respectively). Among cows, a total of 2,369 services were retrieved from 56 herds (263 +/- 8.8 services/sperm dose per sire, range: 233 to 303). Conception rates of cows (29.4%) were not affected by sire or sperm dosage (27.0, 29.1, and 30.3% for the 2.1, 3.5, and 5.0 x 10(6) sperm dosages, respectively). In conclusion, these data indicate that an increased sperm dosage may enhance virgin heifer conception rates for some (but not all) sires, whereas neither sire nor sexed-sperm dosage affected conception rates of lactating cows. Additional studies of sexed-sperm dosage across a larger sampling of bulls are warranted to determine whether and how such a practice can be implemented cost effectively for the benefit of the dairy industry.

Computer-assisted sperm analysis of fresh and frozen-thawed bovine sperm requires proper handling and preparation, and the type of slide used in the assessment is critical if the resultant data are to be useful quality control measurements. In the present study, 4 different slide viewing chambers, a Makler chamber, a clean slide-coverslip, or a 2- or 4-cell chamber Leja slide, were compared with assess their utility in providing reliable measurements of sperm motility variables. A Hamilton-Thorne IVOS Computer-Assisted Semen Analyzer (CASA) was the instrument used to determine sperm measurements utilizing the 4 different chambers. Fifty-eight different freeze batches of bovine semen that had been collected from 47 bulls at 7 sites that sex-sort sperm using Sexing Technologies sorting criteria were incorporated into the trial. Neither the percentage of motile sperm nor the percentage of progressively motile sperm differed for the Makler chamber vs. slide-coverslip comparisons. Similarly, total and progressively motile sperm did not differ between the 2- and 4-cell chambered Leja slides. However, total and progressive motility of sperm determined with the Makler chamber and slide-coverslip were greater (P< 0.0001) than motilities recorded by the 2- or 4-cell chambered Leja slides. Based on the results, the type of viewing chamber can affect the range of sperm motility values when CASA is used for quality control evaluations of thawed, cryopreserved sex-sorted sperm samples.

The gap in fertility between conventional and sex-sorted bovine sperm, known to be on the order of 10 percentage points, has never been bridged, even by increasing the number of sex-sorted sperm per inseminate. A concerted effort in the last few years has resulted in substantial changes in all stages of the sex-sorting process to develop an improved sex-sorted product called SexedULTRA™ (Sexing Technologies, Navasota, TX, USA). In vitro tests show that SexedULTRA™ maintained sperm integrity better than the previous XY method, and field trials were conducted to determine if this translated into improved bovine field fertility. The initial field trial was performed using ejaculates from 8 Holstein bulls located at Select Sires in Plain City (OH, USA). Each ejaculate was split in 2 aliquots that were then processed following the 2 methods (XY method or SexedULTRA™). A total of 6,930 Holstein heifers were inseminated across 41 commercial herds in the United States. Conception rate data were analysed using mixed model ANOVA (JMP 10.0.0; SAS Institute Inc., Cary, NC) with fixed effects of treatment, bull, bull by treatment interaction, and the random effect of herd. Sex-sorted bull sperm following the SexedULTRA™ method resulted in a greater (P < 0.001) conception rate compared with the XY method sperm (45.7 ± 1.7 v. 41.2 ± 1.6%). This is the first report of an improvement in conception rates using sex-sorted bovine semen in a decade since it became commercially available. A second field trial to investigate dose rate effects and compare against conventional semen was performed using 5 dairy bulls located at German Genetics International GmbH in Cloppenburg (Germany). Each ejaculate was split 4 ways, sex-sorted, and frozen in 0.25-mL straws as follows: XY 2.1 million/straw, SexedULTRA™ 2.1 million/straw, SexedULTRA™ 3.0 million/straw, and SexedULTRA™ 4.0 million/straw. A total of 7,855 heifers were inseminated with these sex-sorted straws, whereas 62,398 heifers were inseminated with conventional straws (15 million/straw) produced using contemporary ejaculates from the same bulls. The 56-day nonreturn rate was calculated by sire and treatment combination, and assigned a weight based on the total number of AI for each combination thereof. Table 1 contains the nonreturn rate weighted means and the relative fertility compared to conventional sperm. Data were analysed using a mixed model ANOVA (JMP 10.0.0; SAS Institute Inc.) with treatment and bull as fixed effects. SexedULTRA™ 4.0 sperm presented the greatest (P < 0.001) 56-day nonreturn rate followed by conventional 15.0 (66.73 v. 65.66%); XY 2.1 presented the lowest (P < 0.001) nonreturn rate. This is the first time a dose response effect with sex-sorted bovine sperm and parity in conception rates with conventional semen has been demonstrated. Table 1. Field trial results for number of inseminations, 56-day nonreturn rate (NRR) weighted means, and relative fertility1 compared with conventional sperm

This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials).

Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex-sorted product, branded SexedULTRA™ (Sexing Technologies, Navasota, TX, USA) that retains sperm integrity to improve post-thaw sperm quality, in vitro embryo production, and field fertility compared with the previous XY method. Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (TX, USA). Each ejaculate was divided in 2 aliquots and then processed in 1 of 2 methods (XY method or SexedULTRA™). Post-thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3-h incubation at 37°C using a computer-assisted sperm motility analyzer (Hamilton Thorne IVOS II system, Hamilton Thorne Biosciences, Beverly, MA, USA). Percent intact acrosomes was also estimated after a 3-h incubation. Results were analysed by a mixed model ANOVA with the fixed effect of treatment and random effect of bull. Percent total motile SexedULTRA™ sperm was greater (P < 0.001) than sperm processed following the XY method at 0 (78.8 v. 67.2%) and 3 h (51.0% v. 39.0%) post-thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 (50.7 v. 44.9%) and 3 h (31.5 v. 4.4%) post-thaw in the SexedULTRA™ sperm. Percent intact acrosomes was also greater in SexedULTRA™ sperm compared with the sperm processed following previous method (78.0 v. 64.0%). In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (PA, USA). Five to 10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of 3 straws and a minimum of 800 oocytes per treatment group (ejaculate × treatment) were included in the comparison for development to 8-cell stage (cleavage rate) and to Day 7 blastocyst stage, measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analysed using a mixed model ANOVA with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P < 0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex-sorting process results in better semen quality post-thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex-sorted bovine sperm. Table 1. Results from IVF and embryo culture using frozen-thawed, sex-sorted semen processed using the XY or the SexedULTRA™ method